The Physarum polycephalum php gene encodes a unique cold-adapted serine-carboxyl peptidase, physarolisin II

Area of Science:

• Biochemistry and Molecular Biology
• Enzymology
• Microbial Genetics

Background:

• The php gene in Physarum polycephalum is transcriptionally active and encodes a protein homologous to serine-carboxyl peptidases.
• Previous research identified physarolisin I, a related enzyme from the same organism.

Purpose of the Study:

• To express and characterize the protein encoded by the php gene.
• To determine the enzymatic activity, substrate specificity, and optimal conditions for the novel enzyme.

Main Methods:

• Expression of the php gene product in Escherichia coli.
• Biochemical assays to determine endopeptidase activity and substrate specificity.
• Enzyme activity and stability measurements at various temperatures.
• Circular dichroism spectroscopy to assess conformational changes.

Main Results:

• The expressed protein, named physarolisin II, demonstrated endopeptidase activity with unique substrate specificity.
• Physarolisin II is a cold-adapted enzyme, with optimal activity between 16-22°C.
• The enzyme is unstable at its optimal temperature due to rapid autolysis (t½ ≈ 5 min at 18°C), but more stable at higher temperatures with reduced activity.

Conclusions:

• Physarolisin II represents a novel serine-carboxyl peptidase from Physarum polycephalum with distinct enzymatic properties.
• Its cold-adapted nature and rapid autolysis present unique characteristics for biochemical study.
• Temperature influences both the activity and stability of physarolisin II, highlighting a complex kinetic profile.